Several methods for isolating nucleic acid from various sources are well known. Early methods employed organic solvents, such as phenol and/or chloroform, to selectively precipitate and then remove proteins from a nucleic acid containing solution. Once the protein was removed, dissolved nucleic acid then could be precipitated using alcohol and collected on a solid surface. An appropriate buffer then was used to solubilize the nucleic acid and thereby remove it from the solid surface.
As previously mentioned, early methods for purifying nucleic acid sequences typically employed organic solvents to differentially precipitate nucleic acid sequences from proteins and other undesired matter found in a source material. Once precipitated, the nucleic acid is easily collected on solid a substrate such as a glass stir rod before it is solubilized in a purified state. The affinity nucleic acid displays for solid substrates in the presence of a chaotropic agent has also been exploited to purify nucleic acid. These sample prep methods in addition to employing chaotropic agents typically use organic solvents, such as an alcohol, to assure that the nucleic acid binds the solid substrate or stays bound to the substrate during washing procedures. While such procedures use relatively low concentrations of organic solvents, in comparison to early methods of isolating nucleic acid where organic solvents were the only reagents employed, the alcohol concentrations used in these procedures nevertheless give rise to significant disposal and safety concerns especially when high volumes of samples are processed.
With the advent of nucleic acid amplification reactions such as, for example, the polymerase chain reaction (PCR), the ligase chain reaction (LCR), and other similar procedures designed to synthesize multiple copies of a target nucleic acid sequence, isolating nucleic acid sequences from source materials (variously referred to as “sample preparation” or “sample prep”) has become an increasingly important research area. Several considerations, outside of the mere purification of nucleic acid sequences, make discovery of useful sample prep methods challenging. For example, sample-to-sample contamination with extraneous nucleic acid is a well documented and significant concern. Additionally, initial samples that contain the desired nucleic acid sequence (or “target nucleic acid sequence”), often times contain very small concentrations of the target sequence, as well as comparatively large concentrations of extraneous nucleic acid. Moreover, sample prep often times is performed in areas that are highly regulated in terms of the reagents that can be used and ultimately discarded. Further, in instances where the nucleic acid is being purified for purposes of use in an amplification reaction, it is important for the nucleic acid to ultimately reside in a buffer that does not comprise components that inhibit enzymes commonly employed in amplification reactions. Hence, several considerations, beyond the mere purification of nucleic acid sequences, must be accounted for in the design of a useful sample prep method.
Thus, there is a need for a sample prep method that provides for quantitative isolation of nucleic acid with minimal handling and does not need flammable organic solvents.